Summary of Study ST002578

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001663. The data can be accessed directly via it's Project DOI: 10.21228/M8R72W This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002578
Study Title	Steady State Metabolomics Study in SMARCA4 mutant(BIN-67) cells cultured in the absence of glucose or glutamine
Study Summary	steady-state abundances of all TCA cycle intermediates were more severely reduced in BIN-67 cells deprived of glutamine than those deprived of glucose
Institute	McGill University
Department	Biochemistry
Laboratory	Sidong Huang Lab
Last Name	Fu
First Name	Zheng
Address	McIntyre Medical Sciences Building, 3655 promenade Sir-William-Osler
Email	zheng.fu2@mail.mcgill.ca
Phone	5143985446
Submit Date	2023-04-24
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	GC-MS
Release Date	2023-05-22
Release Version	1

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Project:

Project ID:	PR001663
Project DOI:	doi: 10.21228/M8R72W
Project Title:	Alanine supplementation exploits glutamine dependency induced by SMARCA4/2-loss
Project Summary:	SMARCA4 (BRG1) and SMARCA2 (BRM) are the two paralogous ATPases of the SWI/SNF chromatin remodeling complexes frequently inactivated in cancers.we uncover that SMARCA4/2-loss represses expression of the glucose transporter GLUT1, causing reduced glucose uptake and glycolysis accompanied with increased dependency on oxidative phosphorylation (OXPHOS); adapting to this, these SMARCA4/2-deficient cells rely on elevated SLC38A2, an amino acid transporter, to increase glutamine import for fueling OXPHOS. Consequently, SMARCA4/2-deficient cells and tumors are highly sensitive to inhibitors targeting OXPHOS or glutamine metabolism. Furthermore, supplementation of alanine, also imported by SLC38A2, restricts glutamine uptake through competition and selectively induces death in SMARCA4/2-deficient cancer cells. At a clinically relevant dose, alanine supplementation synergizes with OXPHOS inhibition or conventional chemotherapy eliciting marked antitumor activity in patient-derived xenografts. Our findings reveal multiple druggable vulnerabilities of SMARCA4/2-loss exploiting a GLUT1/SLC38A2-mediated metabolic shift. Particularly, unlike dietary deprivation approaches, alanine supplementation can be readily applied to current regimens for better treatment of these aggressive cancers.
Institute:	McGill University
Department:	Biochemistry
Laboratory:	Sidong Huang Lab
Last Name:	Fu
First Name:	Zheng
Address:	McIntyre Medical Sciences Building
Email:	zheng.fu2@mail.mcgill.ca
Phone:	5145869072

Subject:

Subject ID:	SU002680
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA258813	Bin67_No Glucose_4_1:25 dilution	Glucose depletion 24h
SA258814	Bin67_No Glucose_2_1:25 dilution	Glucose depletion 24h
SA258815	Bin67_No Glucose_1_SIM	Glucose depletion 24h
SA258816	Bin67_No Glucose_3_SIM	Glucose depletion 24h
SA258817	Bin67_No Glucose_4_SIM	Glucose depletion 24h
SA258818	Bin67_No Glucose_1_1:25 dilution	Glucose depletion 24h
SA258819	Bin67_No Glucose_2_SIM	Glucose depletion 24h
SA258820	Bin67_No Glucose_3_1:25 dilution	Glucose depletion 24h
SA258821	Bin67_No Glucose_4	Glucose depletion 24h
SA258822	Bin67_No Glucose_3	Glucose depletion 24h
SA258823	Bin67_No Glucose_2	Glucose depletion 24h
SA258824	Bin67_No Glucose_1	Glucose depletion 24h
SA258825	Bin67_No Glutamine_2	Glutamine depletion 24h
SA258826	Bin67_No Glutamine_4_SIM	Glutamine depletion 24h
SA258827	Bin67_No Glutamine_3_SIM	Glutamine depletion 24h
SA258828	Bin67_No Glutamine_4_1:25 dilution	Glutamine depletion 24h
SA258829	Bin67_No Glutamine_2_1:25 dilution	Glutamine depletion 24h
SA258830	Bin67_No Glutamine_2_SIM	Glutamine depletion 24h
SA258831	Bin67_No Glutamine_1_SIM	Glutamine depletion 24h
SA258832	Bin67_No Glutamine_3_1:25 dilution	Glutamine depletion 24h
SA258833	Bin67_No Glutamine_1_1:25 dilution	Glutamine depletion 24h
SA258834	Bin67_No Glutamine_3	Glutamine depletion 24h
SA258835	Bin67_No Glutamine_4	Glutamine depletion 24h
SA258836	Bin67_No Glutamine_1	Glutamine depletion 24h
SA258837	Bin67_ctrl_3	No treatment
SA258838	Bin67_ctrl_2	No treatment
SA258839	Bin67_ctrl_1_1:25 dilution	No treatment
SA258840	Bin67_ctrl_4	No treatment
SA258841	Bin67_ctrl_2_1:25 dilution	No treatment
SA258842	Bin67_ctrl_4_1:25 dilution	No treatment
SA258843	Bin67_ctrl_1_SIM	No treatment
SA258844	Bin67_ctrl_1	No treatment
SA258845	Bin67_ctrl_2_SIM	No treatment
SA258846	Bin67_ctrl_3_SIM	No treatment
SA258847	Bin67_ctrl_4_SIM	No treatment
SA258848	Bin67_ctrl_3_1:25 dilution	No treatment

Showing results 1 to 36 of 36

Showing results 1 to 36 of 36

Collection:

Collection ID:	CO002673
Collection Summary:	BIN-67 cells were plated in RPMI supplemented with 6% dialyzed FBS (Wisent Bio, Cat# 080-950) at ~80% confluency the day before experiments. The next day, BIN-67 cells were cultured in the absence of glucose or glutamine for 24h. Cells were washed twice in cold saline solution (NaCl, 0.9 g/l) and metabolites were extracted with 1 ml 80% ice-cold methanol (GC/MS grade).
Sample Type:	Ovarian cancer cells

Treatment:

Treatment ID:	TR002692
Treatment Summary:	BIN-67 cells were plated in RPMI supplemented with 6% dialyzed FBS (Wisent Bio, Cat# 080-950) at ~80% confluency the day before experiments. The next day, BIN-67 cells were cultured in the absence of glucose or glutamine for 24h. Cells were washed twice in cold saline solution (NaCl, 0.9 g/l) and metabolites were extracted with 1 ml 80% ice-cold methanol (GC/MS grade).

Sample Preparation:

Sampleprep ID:	SP002686
Sampleprep Summary:	Cells were washed twice in cold saline solution (NaCl, 0.9 g/l) and metabolites were extracted with 1 ml 80% ice-cold methanol (GC/MS grade). After 2 rounds 10 min sets of sonication (30 seconds on/30 seconds off at high intensity) on slurry ice using a Bioruptor UCD-200 sonicator, the homogenates were centrifuged at 14,000 × g at 4 °C for 10 min. Supernatants were collected and supplemented with internal control (800 ng myristic acid-D27) and dried in a cold vacuum centrifuge (Labconco) overnight. The dried pellets were reconstituted with 30 μL of 10 mg/mL methoxyamine-HCl in pyridine, and incubated for 30 min at room temperature. Samples were then derivatized with MTBSTFA for 30 min at 70 °C. A volume of 1 μL of sample was injected splitless with an inlet temperature of 280 oC into the GC (7890, Agilent)/MS (5975C, Agilent) instrument.

Sampleprep ID:

SP002686

Sampleprep Summary:

Cells were washed twice in cold saline solution (NaCl, 0.9 g/l) and metabolites were extracted with 1 ml 80% ice-cold methanol (GC/MS grade). After 2 rounds 10 min sets of sonication (30 seconds on/30 seconds off at high intensity) on slurry ice using a Bioruptor UCD-200 sonicator, the homogenates were centrifuged at 14,000 × g at 4 °C for 10 min. Supernatants were collected and supplemented with internal control (800 ng myristic acid-D27) and dried in a cold vacuum centrifuge (Labconco) overnight. The dried pellets were reconstituted with 30 μL of 10 mg/mL methoxyamine-HCl in pyridine, and incubated for 30 min at room temperature. Samples were then derivatized with MTBSTFA for 30 min at 70 °C. A volume of 1 μL of sample was injected splitless with an inlet temperature of 280 oC into the GC (7890, Agilent)/MS (5975C, Agilent) instrument.

Combined analysis:

Analysis ID	AN004249
Analysis type	MS
Chromatography type	GC
Chromatography system	Agilent 7890A
Column	Agilent DB5-MS (30m x 0.25mm, 0.25um)
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Agilent 5975C
Ion Mode	POSITIVE
Units	intersity

Chromatography:

Chromatography ID:	CH003155
Instrument Name:	Agilent 7890A
Column Name:	Agilent DB5-MS (30m x 0.25mm, 0.25um)
Column Temperature:	60-320
Flow Gradient:	None
Flow Rate:	0.69 ml/min
Solvent A:	None
Solvent B:	None
Chromatography Type:	GC

MS:

MS ID:	MS003996
Analysis ID:	AN004249
Instrument Name:	Agilent 5975C
Instrument Type:	Single quadrupole
MS Type:	EI
MS Comments:	. A volume of 1 μL of sample was injected splitless with an inlet temperature of 280 oC into the GC (7890, Agilent)/MS (5975C, Agilent) instrument. Metabolites were resolved by separation on DB-5MS+DG (30 m x 250 µm x 0.25 µm) capillary column (Agilent J&W, Santa Clara, CA, USA). Helium was used as the carrier gas with a flow rate such that myristic-d27 acid eluted at approximately 18 min. The quadrupole was set at 150 ˚C, the source was at 230 °C and the GC/MS interface was at 320 ˚C. The oven program started at 60 ˚C held for 1 min, then increased at a rate of 10 ˚C/min until 320 ˚C. Bake-out was at 320 ˚C for 9 min. Metabolites were ionized by electron impact at 70 eV. All samples were injected three times: twice using scan (50-1000 m/z) mode (1x and 25x dilution for steady-state samples or 1x and 24x dilution for tracer samples) and once using selected ion monitoring (SIM) mode. All of the metabolites described in this study were validated against authentic standards confirming mass spectra and retention times. Integration of ion intensities was accomplished using Mass Hunter Quant (Agilent). Generally, M-57+. Ions (and isotopes) were analyzed. Mass isotopomer distribution analysis was carried out using in-house software using an in-house algorithm adapted from Nanchen et al as previously described.
Ion Mode:	POSITIVE